The ability of lead (Pb2+) to impair mental activity and neurodevelopment underscores its role as a pervasive environmental threat. Recently, we have observed that this cation is able to inhibit whole cell and single channel currents activated by N-methyl-D-aspartate (NMDA) in rat cultured hippocampal neurons in a concentration-dependent but voltage-independent manner, without significantly altering currents induced by either quisqualate or kainate. In view of the involvement of NMDA receptors in important physiological processes such as memory and learning, a detailed evaluation of the effect of Pb2+ on these receptors is vital. Furthermore, it is known that the impairment of neurodevelopment and cognitive processes induced by this element is much more severe in children than in adults. Indeed, our preliminary work showed that the effects of Pb2+ on NMDA receptors seem to be much more dramatic at early stages of neuronal development. Thus, several fundamental questions are raised: 1) How does Pb2+ affect the different conductance states of NMDA receptor channels at various stages of neuronal development in culture? 2) What is the site of action of Pb2+ on NMDA receptors? 3) At what age of culture of hippocampal neurons are the NMDA receptors most sensitive to the Pb2+? 4) Do glycine, Zn2+, Ca2+ and Mg2+ affect the actions of Pb2+ on NMDA receptors? 5) How does cell maturation influence the effects of Pb2+ on NMDA receptors? 6) What are the chronic effects of Pb2+ on cell development and NMDA receptor function in hippocampal neurons? To provide the answers to these questions, this project is aimed at studying the electrophysiological responses of cultured rat hippocampal neurons to NMDA in the absence of Pb2+ (control condition), and after acute as well as chronic exposure of the cell culture preparation to different concentrations of the cation. Recordings of whole cell and single channel currents activated by different agonists will be performed on the cultured neurons using standard patch clamp techniques. In addition, a new fast drug perfusion and withdrawal system developed in our laboratory will enable us to make a quantitative kinetic analysis of these currents. The elucidation of the actions of Pb2+ on NMDA receptors should constitute the first and most important step in an attempt to design a rational treatment of its neurotoxic effects in the central nervous system (CNS). As a secondary aim of the proposal, we would like to evaluate the interactions of Pb2+ with neuronal nicotinic acetylcholine receptors (AChR), since there is compelling evidence that in addition to NMDA receptors, AChRs may also play a significant role in cognitive processes. We have already demonstrated that low concentrations of Pb2+ similar to those that block NMDA receptors, markedly depress whole cell currents activated by anatoxin-a and ACh, well known agonists of the AChR. The studies described in this proposal will allow us to determine some of the molecular mechanisms that underlie the complex effects of Pb2+ in the CNS.